TY - JOUR
T1 - Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. I. Analysis of bound metal ions by matrix-assisted UV laser desorption time-of-flight mass spectrometry
AU - Hutchens, T. William
AU - Nelson, Randall W.
AU - Li, Chee Ming
AU - Yip, Tai Tung
N1 - Funding Information:
This project has been funded, in part, with federal funds from the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement number 58-6250-l-003. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
PY - 1992/6/26
Y1 - 1992/6/26
N2 - To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions. Thus, LDTOF-MS is ideally suited for the rapid (3-5 min), sensitive (<1 pmol), accurate (±0.05%), and relatively high resolution (m/Δm = 300-500, full width at half maximum, where m = mass) evaluation of synthetic peptides. Further, LDTOF-MS was found to be an important tool for the characterization of peptide mixtures and peptide-metal ion interactions.
AB - To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions. Thus, LDTOF-MS is ideally suited for the rapid (3-5 min), sensitive (<1 pmol), accurate (±0.05%), and relatively high resolution (m/Δm = 300-500, full width at half maximum, where m = mass) evaluation of synthetic peptides. Further, LDTOF-MS was found to be an important tool for the characterization of peptide mixtures and peptide-metal ion interactions.
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U2 - 10.1016/0021-9673(92)85537-4
DO - 10.1016/0021-9673(92)85537-4
M3 - Article
C2 - 1639921
AN - SCOPUS:0026767248
SN - 0021-9673
VL - 604
SP - 125
EP - 132
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1
ER -